RHAMM is an hyaluronan (HA)-binding protein that is either poorly expressed or not expressed in most normal adult tissues but is highly expressed in aggressive human tumors (Adamia et al. (2005) Curr. Drug Targets Cardiovasc. Haematol. Disord., 5: 3-14; Tammi et al. (2002) J. Biol. Chem. 277: 4581-4584; Toole (2004) Nat. Rev. Cancer, 4: 528-539). RHAMM (gene name HMMR) is the Receptor for Hyaluronic Acid Mediated Motility, also known as CD168. RHAMM is a non-integral cell surface protein (CD168) and an intracellular hyaluronan binding protein. Analyses of animal models suggest roles for RHAMM in tumorigenesis and in other disease processes such as arthritis, consistent with its well-documented in vitro functions in cell migration and proliferation and apoptosis (Turley et al. (2002) J. Biol. Chem., 277: 4589-4592). Although cell migration and proliferation and apoptosis are essential functions for morphogenesis and tissue homeostasis, genetic deletion of RHAMM does not appear to affect embryogenesis or adult homeostasis (Tolg et al. (2003) Oncogene 22: 6873-6882). To date, a primary physiological function for RHAMM has remained elusive.
RHAMM was originally isolated from subconfluent migrating fibroblasts in vitro (Turley (1982) Biochem. Biophys. Res. Commun. 108: 1016-1024) and subsequently cloned from mesenchymal cells (see, e.g., Hardwick et al. (1992) J. Cell Biol., 117: 1343-1350). Since antibodies prepared against a shed form of RHAMM blocked HA-stimulated-fibroblast motility, RHAMM was originally described as a cell surface protein that can transduce motogenic signaling pathways in culture (Turley et al. (2002) J. Biol. Chem. 277: 4589-4592). However, HA-bound RHAMM was later detected in intracellular compartments such as the actin and microtubule cytoskeletons, nucleus and cytoplasm (Adamia et al. (2005) Curr. Drug Targets Cardiovasc. Haematol. Disord., 5: 3-14). More recently, RHAMM has been shown to decorate centrosomes and mitotic spindles. RHAMM appears to be required for mitotic spindle formation in culture and acts on the BRCA1/BARD1 pathway to regulate mitotic spindle integrity (Joukov et al. (2006) Cancer Cell, 127: 539-52). Collectively, these results suggest that RHAMM may have both extracellular and intracellular functions, (Nickel (2005) Traffic 6: 607-614; Samuel et al. (1993) J. Cell Biol., 123: 749-758; Zhang et al. (1998) J. Biol. Chem., 273: 11342-11348) thus resembling a group of proteins including epimorphin/syntaxin-2, and autocrine motility factor/phosphoglucose isomerase that are also found at the cell surface where they transmit signals across the cell membrane even though, like RHAMM, they lack both Golgi-ER export peptides and membrane spanning sequences (Nickel (2005) Traffic 6: 607-614).
Although the intracellular versus extracellular functions of RHAMM have not yet been clearly dissected, accumulating data suggest that both forms may contribute to mesenchymal phenotypes, at least during disease. For example, RHAMM expression in culture is increased in transformed fibroblasts by fibrogenic cytokines such as TGF-β (Samuel et al. (1993) J. Cell Biol., 123: 749-758). Cell surface RHAMM is required for activation through fibrogenic cytokines such as PDGF (Zhang et al. (1998) J. Biol. Chem., 273: 11342-11348). It has also been demonstrated that RHAMM expression is high in clinically aggressive mesenchymal tumors (fibromatoses or desmoid tumors) (see, e.g., Tolg et al. (2003) Oncogene 22: 6873-6882). In a mouse model susceptible to desmoid and upper intestinal tract tumors, genetic deletion of RHAMM strongly reduces desmoid initiation and invasion but not upper intestinal tract tumors. Fibroproliferative processes such as aggressive fibromatosis resemble proliferative/migratory stages of wound healing (Cheon et al. (2002) Proc. Natl. Acad. Sci. USA, 99: 6973-6978). Furthermore, the expression of RHAMM is modulated during wounding (Lovvorn et al. (1998) J. Pediatr. Surg., 33: 1062-1069; discussion 1069-1070).
It has been found that factors that regulate fibroblast function play dual roles in wound repair and tumorigenesis (Bissell (2001) Exp. Mol. Med., 33: 179-190; Park et al. (2000) Mol. Med. Today, 6: 324-329) and mesenchymal stem cell trafficking/differentiation into wound sites has become a topic of study (see, e.g., Fu et al. (2006) Wound Repair Regen., 14: 325-35; Mansilla et al. (2006) Transplant Proc. 38: 967-969; Shumakov (2003) Bull. Exp. Biol. Med., 136: 192-195). Mesenchymal stem cells and resident fibroblasts in wounds have immune-modulatory functions that affect the timing and extent of fibrosis during wound repair (Domaszewska and Oszewski (2006) Ann. Transpint. 11: 45-52).
Genetic loss of RHAMM or blocking RHAMM function using peptides mimicking its hyaluronan binding sequence or antibodies to this sequence have been shown to promote subcutaneous adipogenesis. One such peptide (STMMSRSHKTRSHHV (SEQ ID NO: 1), P-1 peptide, also referred to herein as peptide P15-1) isolated from a random phage library has been shown to bind to hyaluronan, is adipogenic and resembles the hyaluronan binding region of RHAMM, a mesenchymal factor involved in wound repair. Another peptide, peptide B (KLKDENSQLKSEVSK (SEQ ID NO: 2)), which contains several key residues required for an interaction of RHAMM with HA, is strongly adipogenic. It was reported in PCT Publication No. WO 2008/140586 that RHAMM displays an effect in modulation of adipose tissue development. In particular, histology analysis of tissue sections through unwounded skin of RHAMM−/− mice showed that the subcutaneous layer of fat was two to three times thicker than in wild-type littermate skin and fibroblasts grown from RHAMM−/− wounds incorporated high levels of fat droplets and reduced smooth muscle actin. Furthermore, RHAMM−/− dermal fibroblasts converted to adipocytes when grown in adipogenic medium. In contrast fibroblasts grown from litter matched wild-type wounds did not exhibit fat droplets, and expressed abundant smooth muscle actin. RHAMM-rescued dermal fibroblasts do not undergo adipogenic conversion when grown in adipogenic medium. Conversely, image analysis of RHAMM−/− mice showed that they have significantly less visceral fat and a lower bone density than wild-type litter mates. These data were said to indicate that RHAMM has a differential effect on visceral vs. subcutaneous adipogenesis.
It is also believed that RHAMM is selective in its regulation of subcutaneous vs. visceral fat, and that this regulation is associated with effects on bone marrow stem cells since bone density provides a measure of stem cell activity. This indicates that RHAMM affects subcutaneous fat deposition through its ability to regulate mesenchymal stem cell differentiation, a conclusion substantiated by the effect of RHAMM loss on another mesenchymal stem cell type, myofibroblasts. Furthermore, hyaluronan/RHAMM interactions play a role in this effect on mesenchymal differentiation since HA binding peptides also promote adipogenesis. In addition to these in vivo effects, RHAMM−/− dermal fibroblasts spontaneously develop into adipocytes when cultures become crowded while wild type dermal fibroblasts do not.